The invention relates to a DNA vaccine against HIV, which is designed from a clinical primary isolate. One aspect of the invention relates to a method of producing a nucleotide sequence construct, in a prefered aspect based on a cassette system, the nucleotide sequence construct being used as a DNA vaccine. The method can, for example, lead to the disclosed synthetic BX08 HIV-1 envelope vaccine nucleotide sequence construct, designed to generate suitable DNA vaccines against HIV, specifically HIV-1. Furthermore, the invention can be used for the production of recombinant protein antigens.
There is an urgent need for new vaccine strategies against HIV. One such new promising strategy is called genetic immunization or DNA vaccine (Webster et al 1997). Some of the advantages of a DNA vaccine against HIV is the induction of Th cell activation, induction of antibodies also against conformational dependent epitopes, and the induction of cellular immunity. So far, most DNA vaccine envelope genes tried, have been from tissue culture adapted virus strains (Boyer et al 1997) that often differs in several aspects from primary clinical isolates (such as early isolates) e.g. in co-receptor usage (Choe et al 1996, Dragic et al 1997).
One disadvantage in HIV envelope based DNA vaccines may be the intrinsic relatively low expression which is regulated by the Rev expression. This may prevent an optimal investigation of the vaccines in small animal models like mice where Rev is functioning suboptimally. Recently it has been shown using the tissue culture adapted HIV-1 MN strain, that an exchange of the HIV codon usage to that of highly expressed mammalian genes greatly improves the expression in mammalian cell lines and renders the HIV expression Rev independent (Haas et al 1996). Additionally, it is known that rare codons cause pausing of the ribosome, which leads to a failure in completing the nascent polypeptide chain and a uncoupling of transcription and translation. Pausing of the ribosome is thought to lead to exposure of the 3xe2x80x2 end of the mRNA to cellular ribonucleases. The world-wide spread of HIV-1 has presently resulted in 8,500 new infections daily and AIDS is now the number 1 cause of death among US males (and number 3 among US females) aged 25-40 years. The epidemic hot-spots now include Eastern Europe, India and South East Asia and southern Africa. The attempts to solve this world-wide problem involve education, prevention, treatment and vaccine development. Affordable protective vaccines represent the best solution to the world-wide problem of infection with HIV-1. Induction of virus neutralizing antibodies is one of the key components in vaccine development. Several recombinant envelope vaccines have been tested in humans and animals, but they seem unable to induce sufficient protection. In this respect DNA vaccination may provide a different and more natural mode of antigen presentation. It is hoped that the immune responses induced by such DNA vaccines could aid in limiting virus replication, slowing disease progression or preventing occurrence of disease. Unfortunately many HIV envelope vaccines induce only moderate levels of antibodies. This could in part be due to limitations in expression, influenced by regulation by the Rev protein and by a species-specific and biased HIV codon usage. Also the virus variability is considered a barrier for development of antibody based vaccines and thus a tool for more easy producing of closely related vaccine variants is needed.
It has been suggested to improve the immunogenicity and antigenicity of epitopes by certain mutations in the envelope gene. An elimination of certain immune dominant epitopes (like V3) could render less immune dominant but more relevant, conserved, or hidden epitopes more immunogenic (Bryder et al 1999). Also elimination of certain N-linked glycosylation sites could improve the exposure of relevant epitopes and increase the immunogenicity of those epitopes. Thus, it is possible that elimination of the glycosylation sites in V1 and V2 may in a more favourable way expose neutralising epitopes (Kwong et al 1998, Wyatt et al 1998). The HIV envelope contains putative internalization sequences in the intracellular part of gp41 (Sauter et al 1996). Thus it would be relevant to eliminate and/or mutate the internalization signals in a membrane bound HIV envelope vaccine gene to increase the amount of surface exposed vaccine derived HIV glycoproteins as gp150. Since the antibody response, that is measured and calculated in titers, is improved by adding the secreted gp120 as opposed to adding the membrane bound form (Vinner et al 1999), it could be advantageous to express the vaccine as a secreted gp120 or a secreted gp140. This would include important parts of gp41, such as the 2F5 neutralising linear epitope (Mascola et al 1997).
Our suggested solution to the problems described above is to design DNA envelope vaccines from a clinical primary isolate with Rev-independent high expression in mammals, that is-built as a cassette for easy variant vaccine production.
A method of producing a nucleotide sequence construct with codons from highly expressed mammalian proteins based on a cassette system coding for an early, primary HIV envelope is described. The method comprises the steps of direct cloning of an HIV gene, derived from a patient within the first 12 months of infection, thereby obtaining a first nucleotide sequence; designing a second nucleotide sequence utilising the most frequent codons from mammalian highly expressed proteins to encode the same amino acid sequence as the first nucleotide sequence; redesigning the second nucleotide sequence so that restriction enzyme sites surround the regions of the nucleotide sequence encoding functional regions of the amino acid sequence and so that selected restriction enzyme sites are removed, thereby obtaining a third nucleotide sequence encoding the same amino acid sequence as the first and the second nucleotide sequence; redesigning the third nucleotide sequence so that the terminals contain convenient restriction enzyme sites for cloning into an expression vehicle; producing snuts between restriction enzyme sites as well as terminal snuts and introducing snuts into an expression vehicle by ligation. The nucleotide sequence construct obtained by this method uses the mammalian highly expressed codons (FIG. 1) and renders the envelope gene expression Rev independent and allows easy cassette exchange of regions surrounded by restriction enzyme sites that are important for immunogenicity, function, and expression.
The method can, for example, lead to the disclosed synthetic, Rev-independent, clinical (such as early), primary HIV-1 envelope vaccine gene, built as a multi cassette. From the sequence of the envelope of the HIV-1 BX08 isolate (personal communication from Marc Girard, Institute Pasteur, Paris), the present inventors have designed a synthetic BX08 HIV-1 envelope vaccine nucleotide sequence construct.
With the great diversity of envelopes in HIV among different patients and within one patient, it would be of advantage to vaccinate with several envelope variants, all being highly expressed. To avoid synthesising several full length envelopes, it is much easier to exchange relevant parts of an envelope cassette to various strains in a multivalent vaccine.
Whether it is the disclosed synthetic BX08 nucleotide sequence construct, or any of the nucleotide sequence constructs obtained by the method, they are designed to generate suitable DNA vaccines against HIV, specifically HIV-1. In this case the mammal, preferably a human being, is inoculated with the nucleotide sequence construct in an expression vehicle and constitutes a host for the transcription and translation of the nucleotide sequence construct. The nucleotide sequence constructs of the present invention can furthermore be used for the production of recombinant protein antigens. In this case the nucleotide sequence construct is placed in an expression vehicle and introduced into a system (e.g. a cell-line), allowing production of a recombinant protein with the same amino acid sequence. The recombinant protein is then isolated and administered to the mammal, preferably a human being. The immune system of the mammal will then direct antibodies against epitopes on the recombinant protein. The mammal, preferably a human being, can thus be primed or boosted with DNA and/or recombinant protein obtained by the method of the invention.
A relevant HIV DNA vaccine can potentially be used not only as a prophylactic vaccine, but also as a therapeutic vaccine in HIV infected patients, e.g. during antiviral therapy. An HIV specific DNA vaccine will have the possibility to induce or re-induce the wanted specific immunity and help the antiviral therapy in limiting or even eliminating the HIV infection. The immunogenicity and antigenicity of epitopes in the envelope can be improved by certain mutations in the envelope gene. The cassette system allows for easy access to the relevant parts of the envelope gene, and thereby eased efforts in the process of genetic manipulation. Some suggested mutations are: an elimination of certain immune dominant epitopes (like V3); elimination of certain N-linked glycosylation sites (like glycosylation sites around V2); elimination and/or mutation of the nucleotide sequence encoding the internalization signals in the cytoplasmic part of a membrane bound HIV envelope to increase the amount of surface exposed vaccine derived HIV glycoproteins; elimination or mutation of the cleavage site between gp120 and gp41; with introduced mutations in gp41 for preserving conformational epitopes.
Table 1 below, lists the nucleotide sequence constructs of the invention by the names used herein, as well as by reference to relevant SEQ ID NOs of DNA sequences, and the amino acid sequence encoded by the DNA sequence in the preferred reading frame. It should be noted, that the snut name consist of the number of the approximate position for the end of the snut and the restriction enzyme used to cleave and/or ligate that end of the snut.
One aspect of the present invention relates to a method for producing a nucleotide sequence construct coding for an HIV gene. The nucleotide sequence construct is produced as a cassette system consisting of snuts. A snut (S) is a nucleotide sequences construct between restriction enzyme cleavage sites comprising the minimal entity of the cassette system. First an HIV gene is obtained from a patient within the first 12 months of infection. The term HIV should be understood in the broadest sense and include HIV 1 and HIV 2. It is possible to determine the period in which the infection has taken place with an accuracy depending on the frequency of the blood tests taken from the patient. For example, patients suffering from various diseases such as lack of certain factors in their blood or hepatitis have their blood tested on a regular basis making it possible to determine the period in which the infection has taken place. Apart from patients with diseases wherein blood tests are used to monitor the course of the disease, other groups of patients have blood tests taken, e.g. blood donors. Unfortunately, humans are still infected due to transfer of virus in blood samples, medical equipment, etc., making it possible to determine the date where the infection has taken place within the time frame of a few days. The importance of obtaining the virus early in the course of the infection is due to the known fact that many early isolates share the common feature of staying relatively constant in their envelope sequences (Karlsson et al., 1998). As these early isolates may share cross-reactive antibody- and/or T-cell epitopes a vaccine based on such early isolates would have a better chance of inducing immune response to shared epitopes of the virus. It is believed that an early, directly cloned virus isolate will share immunogenic sites with other early virus isolates seen during an HIV infection, so that if a mammal generates antibodies and/or T-cells directed against these epitopes, the transferred virus will be eliminated prior to the extensive mutations that may occur after approximately 12 months of infection. Thus, the virus should be isolated as early as possible, that is within the first 12 months of infection, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 month after infection.
The HIV gene for genetic vaccine is preferably cloned directly from viral RNA or from proviral DNA. Direct cloning in this application stands for the virus not being multiplied in stable cell lines in vitro. It is presently expected that passing the virus through a stable cell line will promote mutation in the virus gene. It is particularly preferred not to pass the virus through cells lines selecting for viruses with CXCR4 receptor usage. Direct cloning also includes multiplication of virus in e.g. PBMC (peripheral blood mononuclear cells) since all virus can multiply in PBMC, and this type of multiplication generally does not select for CXCR4 receptor usage. Multiplication of virus is often necessary prior to cloning. Preferably cloning is performed directly on samples from the patient. In one embodiment of the invention, cloning is performed from patient serum. The cloning is then performed directly on the HIV virus, that is on RNA. In another embodiment of the invention cloning, is performed from infected cells. The cloning is then performed on HIV virus incorporated in the genes in an infected cell (e.g. a lymphocyte), that is on DNA. In the latter case the virus might be a silent virus, that is a non-replicating virus. To evaluate if the virus is silent, capability of multiplication in e.g. PBMC is tested.
Cloning is a technique well known to a person skilled in the art. A first nucleotide sequence is hereby obtained. In another aspect of the invention, the first nucleotide sequence, sharing the properties mentioned with direct cloning, is obtained by other means. This could be from a database of primary isolates or the like.
Based on the first nucleotide sequence, the amino acid sequence encoded by said nucleotide sequence is determined. A second nucleotide sequence encoding the same amino acid sequence is then designed utilizing the most frequent codons from highly expressed proteins in mammalians (see FIG. 1, for example, presenting the most frequent codons from highly expressed proteins in humans).
Presently, it appears that the usage of the most frequent codons from mammalian highly expressed proteins has two advantages: 1) the expression is Rev independent; 2) the level of expression is high. The Rev independence is especially advantageous when performing experiments in mice where the Rev systems is functioning sub-optimally. For the use in human vaccine, Rev independence and high expression are important to increase the amount of antigen produced. The determination of the codons for high expression is in this context based on the statistics from human highly expressed proteins (Haas, Park and Seed, 1996, hereby incorporated by reference). It is contemplated that the expression of a protein can be even higher, when current research in binding between codon (on the mRNA) and anticodon (on the tRNA) reveals codons with optimal binding capabilities, and when interactions in-between codons and/or in-between anticodons are known.
The second nucleotide sequence designed utilizing optimized codons is then redesigned to obtain a third nucleotide sequence. The purpose of the redesigning is to create unique restriction enzyme sites around the nucleotide sequence encoding functional regions of the amino acid sequence. By having unique restriction enzyme sites around the nucleotide sequence encoding functional regions of the amino acid sequence, the nucleotide sequence encoding functional regions of the amino acid sequence can easily be isolated, changed, and re-inserted. Examples of functional regions of the amino acid sequence are transmembrane spanning regions, immunodominant regions, regions with antibody cross reacting domains, fusion domains and other regions important for immunogenicity and expression such as variable region 1 (V1), variable region 2 (V2), variable region 3 (V3), variable region 4 (V4) and variable region 5 (V5).
It is important to select the restriction enzymes sites with care. By changing the second nucleotide sequence to insert restriction enzyme sites around the nucleotide sequence encoding functional regions of the amino acid sequence, the third nucleotide sequence must still code for the same amino acid sequence as the second and first nucleotide sequence do. Thus, if necessary, the second nucleotide sequence is redesigned by changing from optimized codons to less optimal codons. It is understood, that the restriction enzyme sites around the nucleotide sequence encoding functional regions of the amino acid sequence should preferably be placed in the terminal region of the nucleotide sequence encoding functional regions of the amino acid sequence. That is preferably outside the nucleotide sequence encoding functional regions of the amino acid sequence, such as 90 nucleotides away, e.g. 81, 72, 63, 54, 45, 36, 27, 21, 18, 15, 12, 9, 6, 3 nucleotides away, but could also be inside the nucleotide sequence encoding functional regions of the amino acid sequence, such as 54, 45, 36, 27, 21, 18, 15, 12, 9, 6, 3 nucleotides inside the nucleotide sequence encoding the functional region of the amino acid sequence.
The type of restriction enzyme sites allowed is determined by the choice of expression vector. In certain cases, the number of restriction enzyme sites is limited and it is hard, if not impossible, to place unique restriction enzyme sites around all the nucleotide sequences coding for functional regions of the amino acid sequence. This problem can be solved by dividing the entire nucleotide sequence into pieces, so that each piece comprises only unique restriction enzyme sites. Modifications to each of the piece is performed separately prior to assembly of the pieces. It is preferred that the nucleotide sequence is divided into 9 pieces. In another aspect, the nucleotide sequence is divided into 8 pieces, or 7, or 6, or 5, or 4, or 3, or 2 pieces. It is especially preferred that the nucleotide sequence is divided into 3 pieces.
Thus, the redesign of the second nucleotide sequence is an interaction between the choice of cloning vector, expression vector, selection of restriction enzyme sites, dividing into pieces, and exchange of codons to insert restriction enzyme sites. In a preferred embodiment of the present invention the cloning vector is Bluescript allowing the restriction enzyme sites chosen from the group consisting of: EagI, MluI, EcoRV, PstI, ClaI, EcoRI, XbaI, SacI, SpeI, XhoI, HindIII, SacII, NotI, BamHI, SmaI, SaII, DraI, KpnI. If other cloning vectors are chosen, other restriction enzyme sites will be available as known by the person skilled in the art.
As a part of the redesigning of the second nucleotide sequence, selected restriction enzyme sites may be removed. The selected restriction enzyme sites to be removed are those sites that are sites of the same type as the ones already chosen above and that are placed within the same piece. The removal of these restriction enzyme sites is performed by changing from optimized codons to less optimal codons, maintaining codons for the same amino acid sequence.
The third nucleotide sequence is redesigned so that the terminal snuts contain convenient restriction enzyme sites for cloning into an expression vehicle. The expression xe2x80x9cvehiclexe2x80x9d means any nucleotide molecule e.g. a DNA molecule, derived e.g. from a plasmid, bacteriophage, or mammalian or insect virus, into which fragments of nucleic acid may be inserted or cloned. An expression vehicle will contain one or more unique restriction enzyme sites and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is produced. The expression vehicle is an autonomous element capable of directing the synthesis of a protein. Examples of expression vehicles are mammalian plasmids and viruses, tag containing vectors and viral vectors such as adenovirus, vaccinia ankara, adenoassociated virus, cannarypox virus, simliki forest virus (sfv), Modified Vaccinia Virus Ankara (MVA), and simbis virus. In one embodiment of the invention, the expression vector contains tag sequences. In another embodiment of the invention a bacteria is transformed with an expression plasmid vector and the bacteria is then delivered to the patient. Preferred expression vehicles are simliki forest virus (sfv), adenovirus and Modified Vaccinia Virus Ankara (MVA).
The snuts are produced by techniques well known by the person skilled in the art. The preferred method for synthesizing snuts, is herein referred to as xe2x80x9cthe minigene approachxe2x80x9d wherein complementary nucleotide strands are synthesized with specific overhanging sequences for annealing and subsequent ligation into a vector. This can be performed with two sets of complementary nucleotide strands, or with three sets of complementary nucleotide strands. The minigene approach minimises the known PCR errors of mismatches and/or deletions, which may occur due to hairpins in a GC rich gene with mammalian highly expressed codons. In FIGS. 10-21, the production of a representative selection of snuts is illustrated.
For the production of long snuts, that is snuts of more than about 240 nucleotides, the technique of overlapping PCR is preferred as illustrated in FIG. 8. Herein two nucleotide strands about 130 nucleotides long with an overlap are filled to obtain a double strand, which is subsequently amplified by PCR.
For the production of multiple snuts with a length of less than about 210 nucleotides, one preferred technique is normal PCR. In a preferred production technique the snuts are synthesized with the same 5xe2x80x2 flanking sequences and with the same 3xe2x80x2 flanking sequences, as illustrated in FIG. 9. The advantages of this approach is, that the same PCR primer set can be used for amplification of several different snuts.
As known by the person skilled in the art, special conditions have to be used for each individual PCR reaction and it should be optimized to avoid inherent problems like deletions mismatches when amplifying GC rich genes from synthetic ssDNA material. Whichever of the above-mentioned techniques are used, it is well known by the person skilled in the art, that it will be necessary to correct unavoidable mismatches produced either due to the nucleotide strand synthesis material and/or the PCR reaction. This can be performed by site directed mutagenesis techniques.
After the various snuts have been produced, they are assembled into pieces and subsequently into the complete gene. Methods for assembly (such as ligation) are well known by the person skilled in the art.
In a preferred embodiment of the present invention the HIV gene encodes the entire HIV envelope. It is understood that the HIV envelope can be the full length envelope gp160 as well as shorter versions such as gp150, gp140, and gp120 with or without parts of gp41.
As will be known by the person skilled in the art, the HIV is divided into several groups. These groups presently include group M, group O, and group N. Further, the HIV is divided into subtypes A, B, C, D, E, F, G, H, I, and J. In the present invention subtype B is preferred due to the high prevalence of this subtype in the Western countries.
The determination of groups and subtypes is based on the degree of nucleotide sequence identity in the envelope gene is presently defined as follows: If the sequence identity is more than 90% the viruses belong to the same subtype; If the sequence identity is between 80% and 90% the viruses belong to the same group. If the sequence identity is less than 80% the viruses are considered as belonging to different groups.
One aspect of the invention relates to a nucleotide sequence construct in isolated form which has a nucleotide sequence with the general formula (I), (II), (III), or (IV)
P1-S495ClaI-S650-720EcoRI-P2-S1265gp120xe2x80x83xe2x80x83(I)
P1-S495ClaI-S650-720EcoRI-P2-S1265XhoI-S1465PstI-P4gp140xe2x80x83xe2x80x83(II)
P1-S495ClaI-S650-720EcoRI-P2-S1265XhoI-S1465PstI-P4gp150xe2x80x83xe2x80x83(III)
P1-S495ClaI-S650-720EcoRI-P2-S1265XhoI-S1465PstI-P4gp160-S2060SacII-P5xe2x80x83xe2x80x83(IV)
wherein P1 designates the nucleotide sequence SEQ ID NO:41, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 90% thereto;
wherein S495Cial designates the nucleotide sequence SEQ ID NO: 7, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 95% thereto;
wherein S650-720EcoRI designates the nucleotide sequence SEQ ID NO: 9, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 95% thereto;
wherein P2 designates the nucleotide sequence SEQ ID NO: 43, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 85% thereto;
wherein S1265gp120 designates the nucleotide sequence SEQ ID NO: 19, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 70% thereto;
wherein S1265XhoI designates the nuclebtide sequence SEQ ID NO: 17, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 80% thereto;
wherein S1465PstI designates the nucleotide sequence SEQ ID NO: 23, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 90% thereto;
wherein P4gp140 designates the nucleotide sequence SEQ ID NO: 57, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 85% thereto;
wherein P4gp150 designates the nucleotide sequence SEQ ID NO: 55, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 85% thereto;
wherein P4gp160 designates the nucleotide sequence SEQ ID NO: 53, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 85% thereto;
wherein S2060SacII designates the nucleotide sequence SEQ ID NO: 33, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 98% thereto; and
wherein P5 designates the nucleotide sequence SEQ ID NO: 59, a nucleotide sequence complementary thereto, or a nucleotide sequence with a sequence identity of at least 85% thereto.
The design of the parent synthetic BX08 gp160 envelope cassette gene with its variant length genes gp150, gp140, gp120 is outlined in FIG. 2.
The nucleotide sequence construct with the formula (I)
P1-S495ClaI-S650-720EcoRI-P2-S1265gp120xe2x80x83xe2x80x83(I)
(visualized in FIG. 3) (SEQ ID NO: 73) codes for the amino acid sequence of gp120 (SEQ ID NO: 74). This amino acid sequence is the part of the HIV envelope that is secreted. Thus, it contains the immunogenic epitopes without being bound to the cell membrane. This is of particular advantage if the nucleotide sequence construct is used for production of recombinant antigens or for a DNA vaccine as the antibody immune response may be higher to secreted versus membrane bound HIV antigens.
The nucleotide sequence construct with the formula (II)
P1-S495ClaI-S650-720EcoRI-P2-S1265XhoI-S1465PstI-P4gp140xe2x80x83xe2x80x83(II)
(visualized in FIG. 4) (SEQ ID NO: 67) codes for the amino acid sequence of gp140 (SEQ ID NO: 68). This amino acid sequence encodes the gp120 and the extracellular part of the gp41 protein. The amino acid sequence is secreted due to the lack of the transmembrane spanning region. This is of particular advantage if the nucleotide sequence construct is used for production of recombinant antigens as the immunogenic and/or antigenic epitopes in the extracellular part of gp41 are included and is of particular advantage for a DNA vaccine as the antibody immune response may be higher to secreted gp120 versus membrane bound HIV antigens.
The nucleotide sequence construct with the formula (III)
P1-S495ClaI-S650-720EcoRI-P2-S1265XhoI-S1465PstI-P4gp150xe2x80x83xe2x80x83(III)
(visualized in FIG. 5) (SEQ ID NO: 69) codes for the amino acid sequence of gp150 (SEQ ID NO: 70). This amino acid sequence contains all of the envelope protein gp160 except the c-terminal tyrosin containing internalization signals in the intracellular part of gp41. The membrane bound surface expression of the amino acid sequence is thereby maintained and enhanced. Mimicking the organization of the native epitope conformation may by expected, making this nucleotide sequence construct of particular advantage if the nucleotide sequence construct is used as a vaccine.
The nucleotide sequence construct with the formula (IV)
P1-S495ClaI-S650-720EcoRI-P2-S1265XhoI-S1465PstI-P4gp160-S2060SacII-P5xe2x80x83xe2x80x83(IV)
(visualized in FIG. 6) (SEQ ID NO: 71) codes for the amino acid sequence of gp160 (SEQ ID NO: 72) i.e. the entire envelope.
The nucleotide sequence construct designated P1 comprises the nucleotide sequence encoding the amino acid sequence in the first variable region (V1) and the amino acid sequence in the second variable region (V2). In one embodiment of the invention the first variable region is surrounded by EcoRV and PstI restriction enzyme sites, and the second variable region is surrounded by PstI and ClaI restriction enzyme sites but as stated above, the choice of restriction enzyme sites can alter.
The nucleotide sequence construct designated S650-720EcoRI comprises the nucleotide sequence encoding the amino acid sequence in the third variable region (V3). In one embodiment of the present invention S650-720EcoRI is characterized by the restriction enzyme sites EcoRI and XbaI in the terminals.
The nucleotide sequence construct designated P2 comprises the nucleotide sequence encoding the amino acid sequence of the fourth variable and constant region (V4 and C4). In one embodiment of the present invention the forth variable region is surrounded by SacI and XhoI restriction enzyme sites.
The nucleotide sequence construct designated S1265gp120 comprises the nucleotide sequence encoding amino acid sequence of the fifth variable and constant region (V5 and C5). S1265gp120 further comprises a nucleotide sequence encoding a C-terminal stop codon.
The nucleotide sequence construct designated P4gp140 comprises the nucleotide sequence encoding amino acid sequence of the transmembrane spanning region. P4gp140 further comprises a nucleotide sequence encoding a C-terminal stop codon prior to the transmembrane spanning region.
The nucleotide sequence construct designated P4gp160 comprises the nucleotide sequence encoding amino acid sequence of the transmembrane spanning region (trans membrane spanning domain: TMD). In a preferred embodiment of the present invention the transmembrane spanning region is surrounded by HindIII and SacII restriction enzyme sites.
The term xe2x80x9csequence identityxe2x80x9d indicates the degree of identity between two amino acid sequences or between two nucleotide sequences calculated by the Wilbur-Lipman alignment method (Wilbur et al, 1983).
The nucleotide sequence constructs with the formula (I), (II), (III), or (IV) illustrates the flexibility in the present invention. By producing a gene with thexe2x80x2described method enables the production of a plethora of antigens with various immunogenic epitopes and various advantages for production and vaccine purposes. To further illustrate the flexibility of the invention, other changes and mutations are suggested below.
In order to improve the immunogenicity of the nucleotide sequence constructs of the invention it is suggested to change the nucleotide sequence such that one or more glycosylation sites are removed in the amino acid sequence. By removal of shielding glycosylations, epitopes are revealed to the immunesystem of the mammal rendering the construct more immunogenic. The increased immunogenicity can be determined by an improved virus neutralization. Changes in the nucleotide sequence such that one or more N-linked glycosylation sites are removed in the amino acid sequence is well known by the person skilled in the art. Potential glycosylation sites are N in the amino acid sequences N-X-T or N-X-S (wherein X is any amino acid besides P). The glycosylation site can be removed by changing N to any amino acid, changing X to a P, or changing T to any amino acid. It is preferred that N is changed to Q by an A to C mutation at the first nucleotide in the codon, and a C to G mutation at the third nucleotide in the codon. This is preferred to increase the GC content in the nucleotide sequence construct. As an alternative N is changed to Q by an A to C mutation at the first nucleotide in the codon, and a C to A mutation at the third nucleotide in the codon. Preferred mutations in the synthetic BX08 envelope gene to remove potential N-linked glycosylation sites in V1 and/or V2 are A307C+C309A and/or A325C+C327G and/or A340C+C342A and/or A385C+C387A and/or A469C+C471A. Examples of such changes is illustrated in SEQ ID NOs: 47, 49, and 51.
For historical reasons the HIVs have been divided into syncytia inducing strains and non syncytia inducing strains. The assay to determine whether a strain is syncytia inducing is described in Verrier et al 1997, hereby incorporated by reference: It is presently known, that viruses utilising the CXCR4 co-receptor are syncytia inducing strains. It is also, at the present, known that the binding site for the CXCR4 involves the third variable region (V3). In a preferred embodiment the nucleotide sequence construct is changed to create a binding site for the CXCR4 co-receptor. It is presently performed in the third variable regions, preferably by the mutation G865C+A866G.
It is well-established that the HIV envelope comprises immunodominant epitopes. An immunodominant epitope is an epitope that most antibodies from the mammal are directed against. The antibodies directed against these immunodominant epitopes may have little effect in elimination of the virus. It is therefore anticipated that modification of the immunodominant epitopes will induce antibodies directed against other parts of the envelope leading to a better elimination and neutralization of the virus. By modification is understood any change in the nucleotide sequence encoding an immunodominant epitope in the amino acid sequence such that said amino acid sequence no longer contains an immunodominant epitope. Thus, modification includes removal of the immunodominant epitope and decrease of immunogenicity performed by mutagenesis. In a preferred embodiment of the present invention an immunodominant epitope in the third variable region (V3) is modified, such as deleted or altered. In a much preferred embodiment the nucleotides 793-897 are deleted. In yet another preferred embodiment of the present invention an immunodominant epitope has been removed from gp41, such as deleted. This is performed in P7 or P8 by elimination of the nucleotides 1654-1710.
It is anticipated that when gp120 is dissociated from gp41 in a vaccine or antigen, two immunodominant epitopes, one on each protein, are exposed and antibodies are directed against these in the mammal. In the infectious virus, gp120 is coiled on top of gp41 and the gp120/gp41 is most likely organized in a trimer, so that these immunodominant epitopes are hidden and therefore less elimination of virus is observed. By removing the cleavage site between gp41 and gp120 a full length gp160, gp150, or gp140 can be obtained with a covalent binding between gp41 and gp120. Removal of the cleavage site between gp41 and gp120 is preferably performed by a mutation at position C1423A. An example of such a mutation is illustrated in the mutation of S1265XhoI (SEQ ID NO: 17) to S1265gp160 (SEQ ID NO: 21).
In order to stabilize the full length gp160, gp150, and gp140 for example when the cleavage site between gp41 and gp120 has been removed as described above, cysteins can be inserted, preferably inside the gp41 helix creating disulphide bounds to stabilise a trimer of gp41s. In a preferred embodiment of the present invention the cysteins are inserted by the mutation 1618:CTCCAGGC:1625 to 1618:TGCTGCGG:1625. An example of such a change is illustrated in SEQ ID NO: 25.
The above mentioned decrease in immunodominant epitopes combined with the increase in immunogenicity of the other epitopes is expected to greatly enhance the efficacy of the nucleotide sequence construct as a vaccine.
During the production of the nucleotide sequence construct, it is convenient to ligate the snuts into pieces. The pieces, as described above, are characterized by their reversible assembly as there are no duplicate restriction enzyme sites. In a preferred embodiment one piece (herein designated P3) contains P1, S495ClaI, S650-720EcoRI, and P2. Another piece (herein designated P8) contains S1265XhoI, S1465PstI, and P4gp160. Yet another piece (herein designated P7) contains S1265XhoI, S1465PstI, P4gp160, S2060SacII, and P5.
One advantage of the present nucleotide sequence construct is the easy access to exchange and alterations in the content and function of the nucleotide sequence and the encoded amino acid sequence. In one embodiment the nucleotide sequence coding for a functional region or parts thereof of the amino acid sequence is repeated. The repeat could be back-to-back or a functional region or parts thereof could be repeated somewhere else in the sequence. Repeated could mean two (one repetition) but could also be three, six, or nine repeats. In a much preferred embodiment the repetition nucleotide sequence codes for amino acids in the third variable region.
In order to improve the protective capabilities of the invention against infections with HIV, one embodiment of the invention relates to the combination of epitopes. The present nucleotide sequence construct allows insertion of one or more new nucleotide sequences isolated from another group and/or subtype of HIV and/or isolated from another patient. Hereby a vaccine or antigen with two or more epitopes from two or more HIVs is obtained. In a preferred embodiment, the V3 is replaced by the new nucleotide sequence. In a much preferred embodiment, the new nucleotide sequence codes for amino acids in the third variable region of a different HIV isolate.
In order to improve the efficacy of the vaccine, aiming at raising cellular immunity, a nucleotide sequence coding for a T-helper cell epitope is included in the nucleotide sequence construct. The nucleotide sequence coding for a T-helper cell epitope or a T-helper cell epitope containing amino acid sequence can be put in anywhere in the nucleotide sequence construct as long as it does not interact with the function of the envelope molecule. However, it is preferably placed in the tail of the nucleotide sequence construct or between the leader sequence and the envelope gene. The T-helper epitopes are preferably selected from core proteins such as P24gag or from a non-HIV pathogen such as virus, bacteria, e.g. BCG antigen 85. For a therapeutic vaccine an HIV helper epitope is preferred since the patient is already primed by the HIV infection. For a prophylactic vaccine, a T-helper cell epitope from a frequently occurring non HIV pathogen such as Hepatitis B, BCG, CMV, EBV is preferred. Also, since the synthetic BX08 envelope genes may contain T-helper cell epitopes in addition to important antibody epitopes, the synthetic BX08 vaccine genes can be mixed with other DNA vaccines to improve the efficacy of the other DNA vaccine.
One aspect of the present invention relates to individualized immunotherapy, wherein the virus from a newly diagnosed patient is directly cloned, the envelope or subunits corresponding to snuts or pieces is produced with highly expressed codons, inserted into any of the nucleotide sequence constructs described above and administered to the patient as a vaccine. Hereby a therapeutic DNA vaccine is obtained, that will help the patient to break immune-tolerance or induce/reinduce an appropriate immune response. In one embodiment the variable regions of the virus are produced with highly expressed codons and exchanged into any of the nucleotide sequence constructs described above.
In one embodiment of the invention, the nucleotide sequence construct as described above satisfies at least one of the following criteria:
a) serum extracted from a Macaque primate which has been immunized by administration of an expression vector containing the nucleotide sequence construct is capable of eliminating SHIV as determined by quantitative PCR and/or virus culturing.
b) serum extracted from a primate which has been immunized by administration of an expression vector containing the nucleotide sequence construct is capable of neutralising HIV-1 BX08 and /or other HIV-1 strains in vitro.
c) serum, extracted from a mouse which has been immunized by administration of an expression vector containing the nucleotide sequence construct four times in intervals of three weeks and boosted after 15 weeks, is capable of decreasing the concentration of HIV-antigen in a culture of HIV, serum or PBMCs by at least 50%. An example of such procudure is shown in example 9.
In one embodiment of the invention, the nucleotide sequence construct of the invention, is used in medicine. That is, it is used as a vaccine, for the production of a recombinant protein, such that the recombinant protein is used as a vaccine, or the nucleotide sequence construct or the recombinant protein is used in a diagnostic composition. Thus, the nucleotide sequence construct of the invention can be used for the manufacture of a vaccine for the prophylactics of infection with HIV in humans.
Intramuscular inoculation of nucleotide constructs, i.e. DNA plasmids encoding proteins have been shown to result in the generation of the encoded protein in situ in muscle cells and dendritic cells. By using cDNA plasmids encoding viral proteins, both antibody and CTL responses were generated, providing homologous and heterologous protection against subsequent challenge with either the homologous or cross-strain reaction, respectively. The standard techniques of molecular biology for preparing and purifying DNA constructs enable the preparation of the DNA therapeutics of this invention. While standard techniques of molecular biology are therefore sufficient for the production of the products of this invention, the specific constructs disclosed hereinprovide novel therapeutics which can produce cross-strain protection, a result heretofore unattainable with standard inactivated whole virus or subunit protein vaccines.
The amount of expressible DNA to be introduced to a vaccine recipient will depend on the strength of the transcription and translation promoters used in the DNA construct, and on the immunogenicity of the expressed gene product. In general, an immunologically or prophylactically effective dose of about 10 xcexcg to 300 xcexcg is administered directly into muscle tissue. Subcutaneous injection, intradermal introduction, impression through the skin, inoculation by gene gun preferably DNA coated gold particles, and other modes of administration such as intraperitoneal, intravenous, peroral, topic, vaginal, rectal, intranasal or by inhalation delivery are also contemplated. It is also contemplated that booster vaccinations are to be provided. It is further contemplated that booster vaccinations with recombinant antigens are to be provided, administered as described above.
The DNA may be naked, that is, unassociated with any proteins, adjuvants or other agents which impact on the recipients immune system. In this case, it is desirable for the DNA to be in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Alternatively, the DNA may be associated with surfactants, liposomes, such as lecithin liposomes or other liposomes, such as ISCOMs, known in the art, as a DNA-liposome mixture, (see for example WO93/24640) or the DNA may be associated with and adjuvant known in the art to boost immune responses, such as a protein or other carrier. Agents which assist in the cellular uptake of DNA, such as, but not limited to, calcium ions, detergents, viral proteins and other transfection facilitating agents may also be used to advantage. These agents are generally referred to as transfection facilitating agents and as pharmaceutically acceptable carriers.
Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used. A wide range of suitable mammalian cells are available from a wide range of sources (e.g. the American Type Culture Collection, Rockland, Md.; also, see Ausubel et al. 1992). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, for example, in Ausubel et al 1992; expression vehicles may be chosen from those provided, for example, in P. H. Pouwels et al. 1985.
In one embodiment of the present invention the protein encoded by the nucleotide sequence construct is produced by introduction into a suitable mammalian cell to create a stably-transfected mammalian cell line capable of producing the recombinant protein. A number of vectors suitable for stable transfection of mammalian cells are available to the public, for exmaple, in Cloning Vectors: a Laboratory Manual (P. H. Pouwels et al. 1985); methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al. 1992.
Standard reference works describing the general principles of recombinant DNA technology include Watson, J. D. et al 1987; Darnell, J. E. et al 1986; Old, R. W. et al, 1981; Maniatis,T. et al 1989; and Ausubel et al. 1992.